Summary

The choroid plexus in the brain is unique because it is a non-neural
secretory tissue. It secretes the cerebrospinal fluid and functions as a
blood-brain barrier, but the precise mechanism of specification of this
non-neural tissue has not yet been determined. Using mouse embryos and
lineage-tracing analysis, we found that the prospective choroid plexus region
initially gives rise to Cajal-Retzius cells, specialized neurons that guide
neuronal migration. Inactivation of the bHLH repressor genes Hes1,
Hes3 and Hes5 upregulated expression of the proneural gene
neurogenin 2 (Ngn2) and prematurely depleted Bmp-expressing
progenitor cells, leading to enhanced formation of Cajal-Retzius cells and
complete loss of choroid plexus epithelial cells. Overexpression of
Ngn2 had similar effects. These data indicate that Hes genes promote
specification of the fate of choroid plexus epithelial cells rather than the
fate of Cajal-Retzius cells by antagonizing Ngn2 in the dorsal
telencephalic midline region, and thus this study has identified a novel role
for bHLH genes in the process of deciding which cells will have a non-neural
versus a neural fate.

INTRODUCTION

The telencephalic hemispheres are formed by bilateral evagination of the
anterior end of the neural tube. The dorsal telencephalon is further
subdivided along the medial-lateral axis into three regions. The most lateral
region becomes cortical neuroepithelium, which later gives rise to neurons and
glial cells of the cerebral cortex. The medial region (the dorsal
telencephalic midline region) is divided into the most medial part, the
choroid plexus epithelium, and an intermediate part, the cortical hem, which
is a major source of Cajal-Retzius cells of the neocortex
(Grove et al., 1998;
Meyer et al., 2002;
Takiguchi-Hayashi et al.,
2004; Yoshida et al.,
2005). Cajal-Retzius cells are distributed in the neocortex and
guide neuronal migration. It has been shown that this medial-lateral
patterning of the dorsal telencephalon is regulated by a combination of
transcription factors and secreted signaling factors. For example, the
homeodomain transcription factors Msx1/2 and Lhx2/Foxg1 (Bf1) are involved in
the development of the choroid plexus and the cortical neuroepithelium,
respectively, whereas secreted factors such as Bmps regulate specification of
the choroid plexus epithelium by inducing Msx1 and repressing
Lhx2/Foxg1 expression
(Bach et al., 2003;
Xuan et al., 1995;
Furuta et al., 1997;
Porter et al., 1997;
Monuki et al., 2001;
Panchision et al., 2001;
Hébert et al., 2002;
Fernandes et al., 2007).

The choroid plexus is unique in the brain, because it is a non-neural
secretory tissue. It produces the cerebrospinal fluid and functions as a
blood-brain barrier. The choroid plexus derives from both epithelial and
mesenchymal components, with the epithelium facing the ventricular lumen. The
choroid plexus epithelial cells are generated from neuroepithelial cells like
other cell types of the central nervous system, such as neurons, astrocytes
and oligodendrocytes (Sturrock,
1979; Thomas and Dziadek,
1993; Awatramani et al.,
2003; Currle et al.,
2005; Hunter and Dymecki,
2007). The role of Bmp signaling in the development of the choroid
plexus has been intensively analyzed. It has been shown that misexpression of
the constitutively active form of Bmp receptors results in an expansion of the
choroid plexus at the expense of the cortical neuroepithelium
(Panchision et al., 2001),
whereas inactivation of the Bmp receptor results in defects of specification
of choroid plexus epithelial cells
(Hébert et al., 2002;
Fernandes et al., 2007). Bmp
signaling induces expression of the homeodomain factors Msx1/2, which are
involved in the development of the dorsal midline region
(Bach et al., 2003;
Hébert et al., 2002).
However, the precise mechanism underlying generation of this non-neural tissue
during the development of the nervous system is, as yet, undetermined.

In this study, we found that the prospective choroid plexus epithelium of
the telencephalon expresses both the proneural bHLH gene neurogenin 2
(Ngn2) and the repressor genes Hes1 and Hes5, and
gives rise to two cell lineages: choroid plexus epithelial cells and
Cajal-Retzius cells. Furthermore, Hes1-, Hes3- and
Hes5-null mutations lead to the upregulation of Ngn2, to a
lack of choroid plexus epithelial cells and to the promotion of Cajal-Retzius
cell differentiation. Overexpression of Ngn2 had similar effects.
These results suggest that Hes and Ngn2 genes
antagonistically regulate the specification of non-neural (choroid plexus)
versus neural (Cajal-Retzius cell) fate in the mouse brain.

Generation of Hes1 floxed mice

The floxed Hes1 targeting vector (see
Fig. 4A) was linearized with
NotI and transfected into TT2 cells, and G418-resistant clones were
selected. Genomic DNA was digested with HindIII and analyzed by
Southern blot using a 0.6 kb HindIII-BamHI fragment as a
5′-probe. Neomycin selection cassette was removed by transient Cre
expression in the targeted TT2 cells. Genotypes were determined by PCR using
the following primers: 5′-CAGCCAGTGTCAACACGACACCGGACAAAC-3′ and
5′-TGCCCTTCGCCTCTTCTCCATGATA-3′. The sizes of PCR products for
floxed and wild-type alleles are 272 bp and 224 bp, respectively.

RESULTS

Lineage analysis of the prospective choroid plexus region

To determine the cell lineage of the prospective choroid plexus epithelium
of the telencephalon, we first examined whether or not neurogenesis occurs in
this region. The homeodomain factor Msx1 is expressed in this region of mouse
embryos from E10.0 to E11.5 and in the choroid plexus epithelium at E12.5
(Fig. 1B,E,F; see
Fig. 3I,Q). At E10.5, subsets
of Msx1+ cells expressed the proneual factor Ngn2
(Fig. 1C-C″, arrowheads),
suggesting that neurogenesis occurs in the prospective choroid plexus region.
In agreement with this notion, some differentiating neurons (DCX+)
were found in this region (Fig.
1D-D″), although they did not express Msx1, suggesting that
Msx1 is downregulated when Ngn2+ cells start neuronal
differentiation. To trace the lineage of Msx1+ cells, we generated
transgenic mice carrying the Msx1 promoter-driven EGFP reporter.
Because EGFP is relatively stable, it can be used as a short-term lineage
tracer that detects cells expressing Msx1 both currently and
previously. As expected, EGFP was specifically expressed in the prospective
choroid plexus region of transgenic mice from E10.0 to E11.5
(Fig. 1E-M″). At E10.0,
96.6±3.8% of EGFP+ cells expressed Msx1, indicating that the
EGFP expression occurred specifically in Msx1+ cells
(Fig. 1E-E″). At E10.5
and E11.5, subsets of EGFP+ cells expressed Ngn2
(Fig. 1H-H″,K-K″,
arrowheads) and the neuronal marker Tuj1
(Fig. 1I-I″,L-L″,
arrowheads), indicating that some Msx1+ cells indeed differentiated
into neurons. Interestingly, p73, a marker of Cajal-Retzius cells, was also
expressed (Fig.
1J-J″,M-M″, arrowheads), suggesting that neurons
formed in the Msx1+ region are Cajal-Retzius cells.

Formation of Cajal-Retzius cells and choroid plexus epithelial cells in
the dorsal telencephalic midline. pEF-EGFP was introduced into the dorsal
telencephalic midline at E9.5 by in utero microelectroporation, and the fate
of EGFP+ cells was examined at the indicated time points. For the
orientation of the planes, see Fig.
1A,A′. (A-F) At E10.5, all EGFP+ cells
resided in the Msx1+ region (A-C). At E11.5, many
EGFP+ cells migrated tangentially into the cortical neuroepitheium
(D,E), and some of them had already reached the marginal zone of the piriform
cortex (D,F). (G-Q) At E12.5, all cells that migrated laterally seemed
to have reached the piriform cortex (G, arrow). Only two regions, the choroid
plexus epithelium (the origin, asterisk) and the piriform cortex (the
destination, arrow) were labeled with EGFP (G). The cells that remained at the
origin expressed transthyretin (Ttr) (H,I,O,P) but not
reelin (Q). The cells that migrated into the piriform cortex expressed
Lhx5 (J,K, adjacent sections), Tuj1 (L,L′) and reelin
(M,M′, arrowheads) but not calretinin (N,N′). di, diencephalon;
tel, telencephalon. Scale bars: 150 μm in A,D; 500 μm in G; 50 μm in
L-Q.

To further analyze the cell lineage of the prospective choroid plexus
region, we introduced pEF-EGFP, which directs EGFP expression under the
control of the elongation factor 1α promoter, into the prospective
choroid plexus region at E9.5 by using an in utero microelectroporation method
(Fukuchi-Shimogori and Grove,
2001; Fukuchi-Shimogori and
Grove, 2003). At E10.5, all EGFP+ cells resided in the
Msx1+ prospective choroid plexus region of the dorsal telencephalic
midline (n=6) (Fig.
2A-C). However, at E11.5, many EGFP+ cells migrated
tangentially into the cortical neuroepitheium, and some of them had already
reached the marginal zone of the piriform cortex (n=6)
(Fig. 2D-F). At E12.5, most of
the cells that migrated laterally seemed to have reached the piriform cortex
(Fig. 2G, arrow), and only two
regions, the choroid plexus epithelium (the origin,
Fig. 2G, asterisk) and the
piriform cortex (the destination, Fig.
2G, arrow), were labeled with EGFP (n=5). This finding
suggests that the electroporated region gives rise to migrating cells around
E11.5 and ceases the formation by E12.5. It has been reported that
Cajal-Retzius cells in the piriform cortex expressed Lhx5
(Yamazaki et al., 2004), and
the EGFP+ cells in this region seemed to express this marker
(Fig. 2J,K). Furthermore, many
of the EGFP+ cells expressed Tuj1
(Fig. 2L,L′) and reelin
(63.5%, n=148) (Fig.
2M,M′, arrowheads) but not calretinin (11.4%,
n=210) (Fig.
2N,N′). These results suggest that the electroporated region
gives rise to Cajal-Retzius cells destined for the piriform cortex around
E11.5. In this experiment, EGFP was not expressed in the hem
(Wnt2b+, Fig.
2H,I), a known source of Cajal-Retzius cells. Furthermore, cell
migration from the electroporated region ceased by E12.5, although the hem is
known to generate migrating Cajal-Retzius cells even after E13.5
(Takiguchi-Hayashi et al.,
2004). These results support the notion that these Cajal-Retzius
cells do not derive from the cortical hem but from the prospective choroid
plexus region. At E12.5, the cells remaining at the origin expressed the
choroid plexus-specific marker transthyretin (Ttr)
(Fig. 2O,P) but not reelin
(Fig. 2Q). These results
suggest that the prospective choroid plexus region
(Msx1+Wnt2b-) gives rise to two
distinct cell types: Cajal-Retzius cells (neural) and the choroid plexus
epithelium (non-neural) around E10.5 to E11.5.

Expression of Hes1 and Hes5 in the dorsal
telencephalic midline. (A) Scheme of a dorsal view of E12.5
forebrain. Coronal sections are made along the broken line. (B) Schemes
of the coronal sections of the dorsomedial telencephalon at E10.5-E12.5.
(C-F) At E10.5, the dorsal telencephalic midline expressed
Bmp4 and Lmx1a (C,D). Hes1 was likewise expressed
in this region as well as in the neighboring diencephalic and telencephalic
neuroepithelium (E). Hes5 was expressed at a lower level in this
region than in the telencephalic neuroepithelium (F). (G-N) At E11.5,
the Bmp4 and Lmx1a expression domain was elongated (G,H).
Msx1 and Ttr were expressed in differentiating choroid
plexus epithelium (I,J), whereas Wnt2b was expressed in the
prospective cortical hem (K). Hes1 expression was gradually
downregulated in Ttr+ cells (L). Although Hes5
expression was upregulated in the telencephalon and the diencephalon, it was
also downregulated in Ttr+ cells (M). Wnt2b+
and Msx1+ domains were clearly separated at this stage (N).
(O-T) At E12.5, the choroid plexus epithelial cells became thin and
cuboidal (R, Ttr+), whereas the cortical hem was still
pseudostratified. Bmp4 and Lmx1a were expressed in both
regions (O,P). Msx1 expression occurred mainly in the choroid plexus
epithelium (Q), whereas Wnt2b expression occurred in the cortical hem
(R). Hes1 and Hes5 expression was almost completely
repressed in the choroid plexus epithelium (S,T). Scale bars: 100 μm in
C-F; 200 μm in G-T.

Expression of Hes1 and Hes5 in the developing
dorsal telencephalic midline region

To reveal the molecular mechanism of the fate choice in the dorsal midline
region, we examined expression of Hes1 and Hes5 from E10.5
to E12.5. The telencephalic choroid plexus forms bilaterally at the
dorsomedial edge of the telencephalon (Fig.
3A). At E10.5, the epithelium of the dorsal telencephalic midline
region expressed Bmp4 and the homeodomain gene Lmx1a, which
regulates development of the choroid plexus and the cortical hem
(Fig. 3C,D)
(Millonig et al., 2000;
Kuwamura et al., 2005).
Likewise, Hes1 was expressed in this region, as well as in the
neighboring diencephalic and telencephalic neuroepithelium, while
Hes5 was expressed at a lower level in this region than in the
telencephalic neuroepithelium at E10.5
(Fig. 3E,F). At E11.5, the
Bmp4 and Lmx1a expression domain was elongated
(Fig. 3G,H) and gradually
divided into two regions, the choroid plexus epithelium
(Msx1+,Ttr+) and the cortical hem
(Wnt2b+) (Fig.
3I-K,N). At this stage, Hes1 and Hes5 expression
continued but was gradually downregulated in Ttr+ cells of
the prospective choroid plexus region (Fig.
3L,M). At E12.5, the choroid plexus epithelial cells became thin
and cuboidal (Ttr+), whereas cells in the cortical hem
were still pseudostratified (Fig.
3R). Bmp4 and Lmx1a were expressed in both
regions, whereas Msx1 and Wnt2b expression occurred in the
choroid plexus epithelium and in the cortical hem, respectively
(Fig. 3O-R). At this stage,
Hes1 and Hes5 expression occurred at a high level in the
cortical hem but was almost completely repressed in the differentiated choroid
plexus epithelium (Fig. 3S,T).
These results show that Hes1 and Hes5 expression occurs in
the prospective choroid plexus region at E10.5 to E11.5, when fate choice
between choroid plexus epithelial cells and Cajal-Retzius cells takes place,
and is downregulated at E12.5, when the cell fate is completely specified.

Generation of Hes1 cKO mice. (A) Strategy for
generation of Hes1 cKO mice. (B) Genomic DNA from
drug-resistant cells was digested with HindIII and analyzed by
Southern blot using a 0.6-kb HindIII-BamHI fragment as a
5′-probe, which detected wild-type and floxed fragments (6.0 kb),
floxed-neo (7.8 kb) and deleted fragments (3.8 kb). (C-E)β
-Galactosidase activity in the forebrain of Emx1-Cre;R26R mice at
E10.5-E12.5. (F-O) Hes1 and Hes5 were expressed in
the dorsal telencephalon of the control (Bmp4+, F,L) at
E10.5 and E11.5 (G,H,M). In Hes1 cKO mice, Hes1 expression
was downregulated around E10.5 (J, asterisk) and was lost by E11.5 (O,
asterisk). Insets of M and O show immunohistochemistry for Hes1. Hes1 protein
expression was lost in Hes1 cKO mice by E11.5. Hes5
expression was upregulated in Hes1 cKO mice (K). di, diencephalon;
tel, telencephalon; hem, cortical hem. Scale bars: 100 μm in C,D,F-O; 200μ
m in E.

Generation of conditional Hes-null mice

The above results suggest that both Hes1 and Hes5 are
expressed in the prospective choroid plexus epithelium when neural versus
non-neural cell fate specification occurs. To reveal the role of Hes genes in
this region, we decided to examine Hes-null mice. However,
Hes1;Hes5 double-null embryos die by E11 before the
establishment of the telencephalon
(Hatakeyama et al., 2004), and
thus they were not suitable for analysis. To overcome this problem, we
generated Hes1 floxed mice, in which the region containing exons 2 to
4 was deleted by Cre recombinase (Fig.
4A,B). These mice were crossed with Emx1-Cre mice, which had
previously been shown to efficiently result in the recombination of floxed
alleles in the dorsal telencephalon
(Iwasato et al., 2000). It has
been reported that expression of Emx1 starts at E9.5
(Yoshida et al., 1997). To
monitor the Cre-mediated recombination, we crossed Emx1-Cre mice with R26R
reporter mice (Soriano, 1999).
Recombination occurred efficiently in the dorsal telencephalic
neuroepithelium, including progenitors to the choroid plexus epithelium at
E10.5 to E12.5 (Fig. 4C-E). We
generated the Hes1 conditional knock-out (cKO) mice by crossing
Hes1 floxed mice and Emx1-Cre mice. In Hes1 cKO mice,
Hes1 expression in the dorsal telencephalon was downregulated around
E10.5 (Fig. 4J, asterisk) and
was lost by E11.5 (Fig. 4O,
asterisk). Thus, compared with the control, where Hes1 expression was
lost by E12.5, downregulation of Hes1 occurred 1-2 days earlier in
Hes1 cKO mice. No apparent defect was observed in the developing
telencephalon of Hes1 cKO mice (data not shown), probably owing to
compensation by other members of the Hes family such as Hes5, which
was upregulated in Hes1 cKO mice
(Fig. 4K, compare with
4H). Additionally,
Hes3 could be upregulated in the absence of Hes1 and
Hes5, and we decided to make mice lacking Hes1, Hes3 and
Hes5. Because Hes3;Hes5 double-null mice are
apparently normal (Hatakeyama et al.,
2004), we generated Hes1 cKO mice on a
Hes3;Hes5 double-null background
(Hes1;Hes3;Hes5 cKO).

Although it has previously been shown that the midbrain, the hindbrain and
the spinal cord can develop severe defects such as premature depletion of
neural progenitors and disruption of the neural tube structures in
conventional Hes1;Hes3;Hes5 KO mice
(Hatakeyama et al., 2004;
Baek et al., 2006), it was
surprising that the cortical hem and the cortical neuroepithelium were only
mildly affected in Hes1;Hes3;Hes5 cKO mice.
Hes1;Hes3;Hes5 cKO mice showed accelerated
differentiation of neurons, including Cajal-Retzius cells in the dorsal
telencephalon (see Fig. S1 in the supplementary material), but there were many
neural progenitors, and the laminar structures of the neocortex and the
hippocampus were not affected (see Fig. S2 in the supplementary material).
Hes-related genes Hey1 and Hey2 were found to be expressed
in the cortical hem and the cortical neuroepithelium (see Fig. S3 in the
supplementary material), and Hey1 expression was upregulated in
Hes1;Hes3;Hes5 cKO mice (see Fig. S3A,B in the
supplementary material). It has previously been shown that both Hey1
and Hey2 inhibit neuronal differentiation and promote maintenance of
neural progenitors, such as Hes genes
(Sakamoto et al., 2003).
Therefore, such mild phenotypes of the dorsal telencephalon of
Hes1;Hes3;Hes5 cKO mice were probably due to
compensation by Hey1 and Hey2. However, Hey1 and
Hey2 were not expressed in the prospective choroid plexus epithelium
(see Fig. S3 in the supplementary material and data not shown), and
Hes1;Hes3;Hes5 cKO mice displayed a severe defect
of the choroid plexus, as described below.

Defect of the choroid plexus and increase of Cajal-Retzius cell
formation in Hes1;Hes3;Hes5 cKO mice

In the control mice, the neuroepithelial cells at the midline became
flattened from E11.5 to E12.5 (Fig.
5A,A′,B,B′), and the thin cuboidal epithelium
protruded into the lateral ventricles around E12.5 to E15.5
(Fig. 5C). By contrast, in
Hes1;Hes3;Hes5 cKO mice, the dorsal midline cells
were not flattened but remained pseudostratified at E11.5 and E12.5
(Fig. 5D,D′,E,E′,
asterisks). This region was morphologically very similar to the neighboring
cortical and diencephalic neuroepithelium. Even at E15.5, no choroid plexus
was formed in Hes1;Hes3;Hes5 cKO mice
(Fig. 5F, asterisk).
Furthermore, expression of the choroid plexus epithelium-specific gene
Ttr was not detectable in the mutant mice at E11.5 or E12.5
(Fig. 5H,J), although it had
already occurred in the control (Fig.
5G,I). These results indicate that the choroid plexus is
completely missing in Hes1;Hes3;Hes5 cKO mice.
Similarly, the choroid plexus in the fourth ventricle was severely affected in
Hes1;Hes5 conventional KO mice at E10.5 (see Fig. S4 in the
supplementary material), although it was not yet formed in the telencephalon
of both the wild-type and Hes1;Hes5 conventional KO mice at
this stage.

Defect of the choroid plexus and increase of Cajal-Retzius cell
formation in Hes1;Hes3;Hes5 cKO mice.
(A-F) HE staining of the dorsal telencephalic midline. In
Hes1;Hes3;Hes5 cKO mice, the dorsal midline cells
were not flattened but remained pseudostratified at E11.5 and E12.5
(D,D′,E,E′, asterisks). Even at E15.5, no choroid plexus was
formed in Hes1;Hes3;Hes5 cKO mice (F, asterisk).
(G-J) Ttr was expressed in the control at E11.5 and E12.5
(G,I) but not in Hes1;Hes3;Hes5 cKO mice (H,J).
(K-N,R-U) Cajal-Retzius cells
(reelin+,Lhx5+) of the mutant piriform cortex
were increased in number (L,N,S,U arrows), compared with the control (K,M,R,T)
at both E11.5 and E12.5. (O-Q) The number of reelin+ cells
was quantified by counting DAPI+ cells on every five sections from
four independent embryos for each genotype. *P<0.01,
t-test. Scale bars: 100 μm in A,D; 200 μm in B,E,G-N; 250 μm
in C,F; 50 μm in R-U.

We then examined the lineage of Cajal-Retzius cells which originate from
the prospective choroid plexus region and migrate into the piriform cortex. We
found that there were more Cajal-Retzius cells
(reelin+,Lhx5+) in the marginal zone of the piriform
cortex of Hes1;Hes3;Hes5 cKO mice than in the
control mice at both E11.5 (Fig.
5K,L,R,S, arrows) and E12.5
(Fig. 5M-Q,T,U, arrows). This
finding suggests that Cajal-Retzius cell development is enhanced in the
absence of Hes genes. Although significant defects were not observed in the
cortical development (see Fig. S2 in the supplementary material), it is
possible that overall acceleration of cortical neurogenesis is involved in
enhancement of Cajal-Retzius cell formation in the piriform cortex of
Hes1;Hes3;Hes5 cKO mice. However, Cajal-Retzius
cell formation was not significantly affected in the pallial-subpallial
boundary region of the mutant mice (see Fig. S5 in the supplementary
material). Furthermore, we generated Hes1;Hes3;Hes5
cKO mice by using Nes-CreERT2 mice
(Imayoshi et al., 2006), in
which Hes1 was knocked out in the cortical neuroepithelium and the
hem but not in the choroid plexus region (see Fig. S6A-F′ in the
supplementary material), which developed normally (see Fig. S6I,L in the
supplementary material). In these mice, Cajal-Retzius cell formation was not
significantly affected in the piriform cortex (see Fig. S6M-P′ in the
supplementary material). These results suggest that inactivation of Hes genes
in the prospective choroid plexus region mainly contributes to enhancement of
Cajal-Retzius cell formation in the piriform cortex, although the possibility
of contribution by overall accelerated neurogenesis is not totally
excluded.

It was previously shown that the telencephalic choroid plexus is missing in
the absence of the Bmp receptor gene Bmpr1a
(Hébert et al., 2002).
We therefore examined expression of Bmp signaling and related molecules in
Hes1;Hes3;Hes5 cKO mice. At E11.5, in these mutant
mice, the expression domain of Bmp4 and Lmx1a was reduced in
size (Fig.
6A.A′,B,B′), and the expression of the downstream
homeodomain genes Msx1 and Msx2 was severely downregulated
compared with the control (Fig.
6C,C′,D,D′). Thus, Bmp signaling was attenuated in the
absence of Hes genes. However, expression of the Bmp receptor Bmpr1a
(see Fig. S7A,B in the supplementary material) and of Noggin, an
antagonist of Bmp (data not shown), as well as its responsiveness to Bmp (see
Fig. S7C-F in the supplementary material) were not affected in
Hes1;Hes3;Hes5 cKO mice. The expression domain of
Wnt3a was also reduced in size at this stage
(Fig. 6E,E′), although
expression of Foxg1 and Lhx2, which are required for
cortical development (Xuan et al.,
1995; Porter et al.,
1997; Monuki et al.,
2001), was not significantly affected
(Fig. 6F,F′,G,G′).
In Hes1;Hes3;Hes5 cKO mice, the dorsal
telencephalic midline was reduced in size, but the cortical neuroepithelium
did not expand. Cell death and proliferation were not responsible for the
reduction in size of the dorsal telencephalic midline (see Fig. S7G-L in the
supplementary material).

At E12.5, in the control mice, the telencephalic midline region was clearly
separated into the choroid plexus epithelium and the cortical hem, while
Bmp4 and Lmx1a were expressed in both regions
(Fig. 6H,I). In
Hes1;Hes3;Hes5 cKO mice, the prospective choroid
plexus region remained pseudostratified, and the Bmp4 and
Lmx1a expression domain became smaller
(Fig. 6H′,I′). In
the control, Msx1 was expressed at a high level in the choroid plexus
epithelium and at a low level in the ventral part of the cortical hem
(Fig. 6J), while Msx2
was expressed in both the choroid plexus epithelium and the cortical hem
(Fig. 6K). In
Hes1;Hes3;Hes5 cKO mice, both Msx1 and
Msx2 were expressed at very low levels
(Fig. 6J′,K′,
asterisks). However, the Wnt3a expression domain was not
significantly changed between control and
Hes1;Hes3;Hes5 cKO mice at this stage
(Fig. 6L,L′). Expression
of the homeodomain gene Lhx5 in the eminentia thalami, which
physically links the telencephalic choroid plexus to the diencephalon
(Hébert et al., 2002),
was not significantly affected in Hes1;Hes3;Hes5
cKO mice, indicating that the diencephalon is not expanded in the absence of
Hes genes (Fig. 6M,M′).
This finding reveals that inactivation of Hes genes leads to attenuation of
Bmp signaling and lack of the choroid plexus epithelium with no expansion of
the cortical and diencephalic neuroepithelium.

Bmp signaling and homeodomain gene expression in
Hes1;Hes3;Hes5 cKO mice. (A-G′) At
E11.5, in Hes1;Hes3;Hes5 cKO mice, the expression
domains of Bmp4, Lmx1a, Msx1, Msx2 and Wnt3a were reduced in
size, compared with the control, although Foxg1 and Lhx2
expression (cortex) was not significantly affected. (H-M′) At
E12.5, in the control, the telencephalic midline region was clearly separated
into the choroid plexus epithelium and the cortical hem, and Bmp4 and
Lmx1a were expressed in both regions (H,I). In
Hes1;Hes3;Hes5 cKO mice, the prospective choroid
plexus region remained pseudostratified, and Bmp4 and Lmx1a
expression domain was smaller in size (H′,I′). Msx1 and
Msx2 expression domains were also reduced in size (J′,K′,
asterisks), whereas expression of Wnt3a (cortical hem) and
Lhx5 (eminentia thalami) was not significantly affected
(L′,M′). Scale bars: 100 μm in A-G′; 200 μm in
H-M′.

Upregulation of proneural genes in the dorsal telencephalic midline
of Hes1;Hes3;Hes5 cKO mice

In Hes1;Hes3;Hes5 cKO mice, Cajal-Retzius cells
increased in number in the piriform cortex at E12.5. Furthermore, neurogenesis
was accelerated in the dorsal telencephalic midline region of
Hes1;Hes3;Hes5 cKO mice at E10.5 and E11.5
(Fig. 7B,D, asterisks) compared
with the control mice (Fig.
7A,C). We then sought to determine the mechanism for this enhanced
Cajal-Retzius cell formation in Hes1;Hes3;Hes5 cKO
mice. In the dorsal telencephalic midline (Lmx1a+) of
wild-type embryos, Ngn1 and Ngn2 were expressed at E10.5 and
E11.5 (Fig. 7G-K).
Interestingly, at E10.5, Hes1 and Ngn2 were co-expressed by many cells
(Fig. 7E, arrowheads), but the
expression became mostly segregated at E11.5
(Fig. 7F, arrows), suggesting
that Hes1+Ngn2+ cells gradually become either
Hes1+ or Ngn2+ cells during this period. In
Hes1;Hes3;Hes5 cKO mice, Ngn1 and
Ngn2 expression were highly upregulated at both E10.5 and E11.5
(Fig. 7L-P, asterisks,
7R,R′,S) compared with the control
(Fig. 7G-K,Q,Q′,S). These
results suggest that inactivation of Hes genes leads to upregulation of
Ngn1 and Ngn2 expression, which contributes to enhanced
Cajal-Retzius cell formation.

To further clarify the role of Ngn2 in Cajal-Retzius cell
formation, we next examined Ngn2-null mice
(Fode et al., 2000). The
number of Cajal-Retzius cells (reelin+, p73+),
which are derived from the dorsal telencephalic midline, was reduced in
Ngn2-null mice compared with the control mice (see Fig. S8 in the
supplementary material), indicating that Ngn2 indeed contributes to
Cajal-Retzius cell formation. Nevertheless, there was no significant
difference in the number of Cajal-Retzius cells in the piriform cortex (data
not shown). It is partly because Cajal-Retzius cells in this region come from
other regions in addition to the choroid plexus region
(Bielle et al., 2005).
Furthermore, the dorsal telencephalic midline region developed normally in
Ngn2-null mice (see Fig. S9 in the supplementary material),
suggesting that Ngn1 compensates Ngn2 to some extent.

We found that in the absence of Hes genes, Ngn2 expression was
upregulated and that Cajal-Retzius cells in the piriform cortex increased in
number at the expense of the choroid plexus cell fate. We then examined
whether misexpression of Ngn2 in the dorsal telencephalic midline
promotes formation of Cajal-Retzius cells at the expense of the choroid
plexus. Misexpression of Ngn2 in the dorsal telencephalic midline at
E9.5 inhibited the development of the choroid plexus
(Fig. 8D′,E′)
(n=4). Furthermore, this misexpression of Ngn2 generated
more Cajal-Retzius cells (reelin+) in the piriform cortex
(Fig. 8F,G). These results
suggest that misexpression of Ngn2 in the dorsal telencephalic
midline at E9.5 promotes formation of Cajal-Retzius cells at the expense of
the choroid plexus.

Upregulation of proneural genes in the dorsal telencephalic midline of
Hes1;Hes3;Hes5 cKO mice. (A-D)
Neurogenesis (Tuj1+) was enhanced in the dorsal telencephalic
midline (brackets) of Hes1;Hes3;Hes5 cKO mice at
E10.5 and E11.5 (B,D, asterisks), compared with the control (A,C).
(E,F) Double immunostaining for Ngn2 and Hes1 in wild-type
embryos. Many cells co-expressed Ngn2 and Hes1 at E10.5 (arrowheads), but the
expression was mostly segregated at E11.5 (arrows). (G-S) In the dorsal
telencephalic midline region (Lmx1a+) of control mice,
Ngn1 and Ngn2 expression occurred at low levels in subsets
of cells at E10.5 (G-I) and was down-regulated at E11.5 (J,K,Q,Q′). By
contrast, Ngn1 and Ngn2 expression was highly upregulated in
Hes1;Hes3;Hes5 cKO mice at both E10.5 and E11.5
(L-P, asterisks; R,R′,S). *P<0.01;
**P<0.001, t-test. Scale bars: 50 μm in
A-D,E1,F1,G-I,L-N; 100 μm in J,K,O,P; 20 μm in Q-R′.

To examine the plasticity of the differentiation competency at a later
stage, we electroporated the Ngn2 vector at E11.5 (this procedure
should induce the ectopic expression around E12). Misexpression of
Ngn2 increased Cajal-Retzius cell formation (reelin+) from
the cortical hem (Wnt2b+,
Fig. 8H,I) but did not affect
choroid plexus formation (Ttr+) at E12.5
(Fig. 8J,K). These results
suggest that the choroid plexus epithelial region loses competency to produce
Cajal-Retzius cells by E12.5.

The above results indicate that Hes-expressing cells and
Ngn2-expressing cells are segregated in the dorsal telencephalic
midline region around E10.5 to E11.5, and that Hes-expressing cells
adopt the choroid plexus fate, whereas Ngn2-expressing cells adopt
Cajal-Retzius cell fate. We then sought to determine the mechanism responsible
for this segregation. The most likely mechanism is Notch-mediated lateral
inhibition: proneural genes such as Ngn2 induce expression of the
Notch ligand, leading to activation of the Notch pathway and to the induction
of Hes1/Hes5 expression in neighboring cells (Kageyama et
al., 2007). We thus examined mice mutant for Rbpj, an essential
effector of Notch signaling (Tanigaki and
Honjo, 2007). However, because conventional Rbpj-null
mice die very early (Oka et al.,
1995), we generated Rbpj cKO mice by crossing floxed
Rbpj mice (Han et al.,
2002) with Emx1-Cre mice. In these cKO mice, although
Hes5 expression was downregulated, Hes1 was still expressed
(see Fig. S10C,D,K,L in the supplementary material), and Ttr
expression occurred normally (see Fig. S10E,M in the supplementary material).
These results indicate that the Notch-Rbpj pathway is not involved in
segregation of Hes- and Ngn2-expressing cells.

DISCUSSION

It has been shown that Cajal-Retzius cells are born at multiple places in
the developing telencephalon, such as the cortical hem, the septum and the
pallial-subpallial boundary
(Takiguchi-Hayashi et al.,
2004; Yoshida et al.,
2005; Bielle et al.,
2005). By lineage-tracing analysis, we found that cells in the
prospective choroid plexus region have potential to give rise to Cajal-Retzius
cells first, and later differentiate into choroid plexus epithelial cells.
Thus, neural (Cajal-Retzius) and non-neural (choroid plexus epithelial) cells
are sequentially born in the dorsal telencephalic midline region. We further
showed that Hes1+Ngn2+ cells gradually become either
Hes1+ or Ngn2+ cells, and that inactivation of Hes1,
Hes3 and Hes5 upregulated expression of Ngn2,
accelerating Cajal-Retzius cell formation at the expense of the choroid plexus
(Fig. 9A). In these mutant
mice, it is likely that almost all cells in the prospective choroid plexus
epithelium adopted Cajal-Retzius cell fate and migrated into the piriform
cortex, because the choroid plexus epithelium is completely lacking. However,
it is also possible that some cells remain as pseudostratified epithelial
cells, and further experiments will be required to resolve this issue.
Similarly, overexpression of Ngn2 enhanced formation of Cajal-Retzius
cells and inhibited differentiation of the choroid plexus. These results
suggest that Hes1+Ngn2+ cells are bi-potent and become
segregated into Hes1-expressing cells that adopt the choroid plexus fate and
Ngn2-expressing cells that adopt Cajal-Retzius cell fate, and that
Hes and Ngn2 antagonistically regulate the non-neural versus
neural fate decision in the dorsal telencephalic midline region
(Fig. 9B). However, it is also
possible that these two cell types derive from two different types of
progenitor cells rather than from bi-potent cells. We were not able to resolve
this issue decisively, because it is technically difficult to perform a clonal
dissociation culture of the E10.5 dorsal telencephalic midline.

It is surprising that the prospective choroid plexus region initially give
rise to Cajal-Retzius cells before differentiating into the choroid plexus
epithelium. Because the hem, a well known source of Cajal-Retzius cells, is
located next to the prospective choroid plexus region, it is possible that
these two regions are not clearly separated at early stages and thus some
cells in the boundary region could contribute to Cajal-Retzius cell formation.
However, around E10.5 to E11.5, neurogenesis occurs widely in the prospective
choroid plexus region and is not restricted to the boundary to the prospective
hem region (Fig. 1).
Furthermore, Cajal-Retzius cell migration from the prospective choroid plexus
region ceases by E12.5 (Fig.
2G), although that from the hem continues even after E13.5
(Takiguchi-Hayashi et al.,
2004). These data support the notion that these Cajal-Retzius
cells derive from the prospective choroid plexus region.

Misexpression of Ngn2 inhibits choroid plexus formation and
enhances Cajal-Retzius cell formation in the dorsal telencephalic midline.
(A-G) pEF-EGFP alone (A-C) or the Ngn2 expression vector
together with pEF-EGFP (D-F) was introduced into the dorsal telencephalic
midline at E9.5 by in utero microelectroporation, and the region was analyzed
at E12.5. Misexpression of Ngn2 inhibited formation of the choroid
plexus (E′, asterisk) and increased the number of Cajal-Retzius cells in
the piriform cortex (F,G). (H-K) Ngn2 was overexpressed in the
prospective choroid plexus and cortical hem regions by electroporation at
E11.5, and the coronal sections were examined at E12.5. Forced expression of
Ngn2 at E11.5 increased Cajal-Retzius cell (reelin+)
formation in the cortical hem (H,I, arrows) but did not affect the choroid
plexus development (J,K). *P<0.05, t-test.
Scale bars: 200 μm in A,B,D,E,H-K; 50 μm in C,F.

Differentiation competency of the dorsal telencephalic midline
cells

In the dorsal telencephalic midline of wild-type mice, Hes1 and
Hes5 expression occurred at high levels until E11.5 but was then
downregulated in the choroid plexus epithelium at E12.5. In our
Hes1;Hes3;Hes5 cKO mice, Hes1 expression
occurred at a lower level at E10.5 and was lost around E11.5. Thus, in
Hes1;Hes3;Hes5 cKO mice, Hes1 expression
was repressed only 1 or 2 days earlier than in the control. Nevertheless, we
found profound defects (loss of the choroid plexus), suggesting that
Hes expression around E10.5 to E11.5 has a crucial role in the
specification of the choroid plexus. At this stage, Hes-expressing
cells and Ngn2-expressing cells seem to be segregated in the dorsal
telencephalic midline region, but after this stage, the cell fates seem to be
determined and are unchangeable. In accordance with this notion, development
of the choroid plexus was severely affected by electroporation of the
Ngn2 vector at E9.5 (expression occurs around E10) but not at E11.5
(expression occurs around E12). These results suggest that the differentiation
competency becomes unchangeable soon after E11.5 in the dorsal telencephalic
midline. The mechanism underlying how segregation of choroid plexus epithelial
cells and Cajal-Retzius cells is regulated is not known. Lateral inhibition
mediated by Notch signaling is a possible mechanism. However, inactivation of
Rbpj, an essential mediator of Notch signaling, neither abolishes
Hes1 expression nor significantly affects the choroid plexus
development, thus suggesting that Notch signaling is not involved in this
process.

Although the choroid plexus was completely missing in
Hes1;Hes3;Hes5 cKO mice, the boundary between the
prospective choroid plexus epithelium and the diencephalon was not affected.
Thus, it is likely that none of the cells in the prospective choroid plexus
epithelium adopted the diencephalic cell fate. This finding suggests that
these prospective choroid plexus epithelial cells do not have the competency
to become cell types other than choroid plexus and Cajal-Retzius cells.

The role of Bmp signaling in the non-neural versus neural cell fate
specification

Our finding that Hes1 expression is not regulated by the
Notch-Rbpj pathway raised another important question: which factors regulate
Hes1 expression in the dorsal telencephalic midline? One of the
candidates is Bmp signaling, because previous studies have shown that
activation of Bmp signaling induces Hes1 expression in cultured cells
(Dahlqvist et al., 2003).
Additionally, our preliminary study also showed that treatment with Bmp leads
to increased Hes1 expression in neural progenitor cultures (I.I.,
T.S., T.O. and R.K., unpublished). Furthermore, Bmp genes are expressed at
high levels in the dorsal telencephalic midline. Thus, Bmp signaling seems to
be important for Hes1 expression in this region. Conversely, Hes
genes are required for maintenance of Bmp signaling, because expression of
Bmp and of its downstream genes is severely downregulated in
Hes1;Hes3;Hes5 cKO mice. Apparently, Cajal-Retzius
cells do not express Bmp, so premature differentiation of these cells
may lead to loss of Bmp expression. We speculate that Hes genes
maintain Bmp-expressing cells by inhibiting Cajal-Retzius cell
formation rather than directly activating Bmp expression.

It has been shown that Bmp signaling is required locally for the
development of the dorsal telencephalic midline but not for the medial-lateral
patterning of the dorsal telencephalon
(Hébert et al., 2002).
Regions where Bmp signaling is inactive seem to become the neural cells
(cortical hem and cortical neuroepithelium), whereas regions with a high Bmp
activity become the non-neural cells (the choroid plexus). This effect of Bmp
signaling is reminiscent of the epidermal versus neural fate specification of
Xenopus. In early Xenopus embryos, Bmp signaling induces
naïve ectoderm to adopt the epidermal fate, whereas anti-Bmp factors such
as noggin and chordin inhibit Bmp signaling and promote the neural fate
specification (Sasai and De Robertis,
1997). It is likely that the Bmp-Hes pathway regulates the choroid
plexus fate, whereas the Bmp antagonist-Ngn pathway regulates the
Cajal-Retzius cell fate (Fig.
9B). A full understanding of this process, however, will require
further analysis, including the functional interaction between bHLH and
homeodomain factors that are required for the choroid plexus formation.

Supplementary material

Acknowledgments

We thank Shigeyoshi Itohara, François Guillemot, Tasuku Honjo,
Phillip Soriano and Yoko Suda for reagents. We also thank Hitoshi Miyachi for
help in generating pMsx1-EGFP mice. This work was supported by the
Grants-in-aid from the Ministry of Education, Culture, Sports, Science and
Technology of Japan, and by the Uehara Memorial Foundation. I.I. was supported
by the 21st Century COE Program of the Ministry of Education, Culture, Sports,
Science and Technology of Japan and by Research Fellowships of the Japanese
Society for the Promotion of Science for Young Scientists.

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